Amp-activated protein kinase activating compounds and uses thereof

ABSTRACT

The present invention relates to pharmaceutical compounds, compositions and methods, especially as they relate to the treatment and/or prevention of conditions such as muscle disorders such as polymyositis, dermatomyositis, muscular dystrophy, myasthenia gravis, amyotrophic lateral sclerosis, rhabdomyolysis, and cardiomyopathy, brain disorders including Alzheimer&#39;s, stroke, cerebral atrophy, traumatic brain injury, and dementia, aging, diabetes, and cancer, using compounds of Formula (I):as described herein, including pharmaceutically acceptable salts thereof and pharmaceutical compositions comprising these compounds.

FIELD OF THE INVENTION

The field of this invention is compounds, pharmaceutical compositionsand methods useful for the treatment of conditions associated withenzymes and pathways regulated by AMP. The compounds, compositions andmethods are useful to treat certain cancers, cardiac conditions,diabetic conditions, obesity, and other conditions for which activationof pathways regulated by AMP is beneficial.

BACKGROUND OF THE INVENTION

Acadesine, which is also referred to as5-amino-1-D-ribofuranosyl-1H-imidazole-4-carboxamide,5-aminoimidazole-4-carboxamide riboside, AICA riboside and AICAR, is anatural substance with CAS RN 2627-69-2 and with the following formula:

When phosphorylated, AICAR becomes acadesine 5′-monophosphate, which isalso called AICAribotide or ZMP, has CAS RN 3031-94-5, which is anatural occurring active metabolite of acadesine, an intermediate in thede novo purine biosynthesis pathway (FIG. 1). ZMP acts in vivo as an AMPmimic. AMP is the monophosphate form of ATP, which is often referred toas the energy currency of a cell: ATP is the most immediate source ofenergy in cells When ATP is used to provide energy to a cell, such aswhen a muscle contracts repeatedly, it forms ADP by loss of onephosphate from the triphosphate group. Cellular machinery, e.g.,adenylate kinase, normally converts two ADP molecules into one ATP andone AMP: the ATP provides energy to the cell, and the AMP can also berecycled to ATP, but its presence is treated as a sign that the cellneeds to make more ATP, see FIG. 2. Many physiological processes otherthan energy supply are also regulated by AMP: a rise in AMP isinterpreted to mean the cell is stressed, as by lack of energy oroxygen. AMP interacts with a number of key intracellular metabolicenzymes. As an indicator of energy stress, AMP directly regulatesseveral vital enzymes within the cell that all function to increase theavailability of ATP and improve the cell's ability to maintain energeticbalance in the future. These include fructose-1,6-bisphosphatase (FBP1),phosphofructokinase-1 (PFK-1), glycogen phosphorylase (GP), andAMP-activated protein kinase (AMPK). Thus, when the level of AMP in acell rises above its typical very low concentration, the cell respondsby boosting pathways that promote recovery, growth, healing, energyutilization, carbohydrate metabolism, enhanced cellular metabolism, andother beneficial stress responses. AMP promotes cell survival actionsthat can be beneficial to a human in many ways: it increasesmitochondria development, inhibits gluconeogenesis, decreasesinflammation, and enhances recycling of damaged proteins.

Work in rodents has demonstrated a vast array of beneficial effects ofAICAR, including attenuation of age-related pathology, cognitive declinein dementia, cancer progression, insulin resistance, and functionalimpairment in several muscular dystrophies. Some of these effects ofAICAR presumably occur in part through AMPK activation. However,numerous studies using cells and mice that do not have functional AMPKshow that AICAR has many effects that are independent of AMPK. Theseeffects are likely due, at least in part, to ZMP's effects on FBP-1,PFK-1, GP, and perhaps other AMP-sensitive enzymes. Thus the inventionprovides methods to use ZMP prodrugs for treating such conditions.

Clinical studies in patients undergoing coronary artery bypass graftsurgery demonstrate that treatment with acadesine before and duringsurgery can reduce early cardiac death and myocardial infarction (cf.e.g.: D. T. Mangano, Journal American Medical Association 1997, vol.277, pp. 325-332). Phase III trials have been carried out withacadesine, indicating that it is safe when administered orally andintravenously. There are patents granted and/or patent applicationspublished which relate to the use of acadesine for: preventing tissuedamage due to decreased blood flow (cf. U.S. Pat. Nos. 4,912,092,5,817,640); treating neurodegenerative conditions (cf. U.S. Pat. No.5,187,162); preventing injury to the central nervous system (cf. U.S.Pat. No. 5,236,908); treating obesity (cf. WO 0193873 A1); treating type2 diabetes (cf. WO 0197816A1) and treating conditions associated withinsulin resistance (cf. WO 0209726 A1). There are patents granted and/orpatent applications published which relate to the use of acadesine5′-monophosphate as flavouring material (cf. U.S. Pat. No. 3,355,301),anticholesteremic/antihyperlipidemic agent (cf. WO 9303734 A1),antiobesity agent (cf. WO 0193874 A1) and antidiabetic agent (cf. WO0197816 A1). There are also reports describing the use of acadesine,acadesine 5′-monophosphate or prodrugs thereof for treating leukemia andlymphoma. US2005/0233987, U.S. Pat. No. 7,560,435. Furthermore, examplesof prodrugs of ZMP and uses thereof are disclosed in WO2020/050935. Mostof the biological activities exhibited by ZMP and acadesine are believedto result from mimicking AMP in biochemical pathways that are regulatedby AMP.

It is known that acadesine is an apoptosis inhibitor in some types ofcells. Thus, for instance, it is known that acadesine inhibitsglucocorticoid-induced apoptosis in quiescent thymocytes, that acadesineinhibits apoptosis caused by serum deprivation in fibroblastsoverproducing fructose 2,6-bis-phosphate, and that acadesine inhibitsceramide-induced apoptosis in primary astrocytes. Therefore, shouldacadesine have any effect on lymphocyte apoptosis, acadesine would beexpected to be an inhibitor of it.

Throughout the literature on acadesine, the free nucleoside is generallyadministered, and relatively large dosages are required for efficacy. Inmost of these studies it is assumed that acadesine is rapidly taken upby cells and phosphorylated by adenosine kinase to the active speciesZMP. A number of references also refer to the fact that acadesine is notpassively transported and relies upon adenosine nucleoside transportersfor cell permeation. If these assumptions about adenosine kinase and thenucleoside transporters are true, then the use of acadesine to producethe active species ZMP has two problems. The first problem is thatadenosine kinase is not expressed in equal amounts in every cell type,and dosing of acadesine will produce vastly different amounts of ZMP indifferent tissues.

The second problem with acadesine is the hydrophilic structure of themolecule. AICAR has very low bioavailability when dosed orally, becausemost of the administered compound is excreted unaltered in urine. Thismakes the preferred method of dosing intravenous injection or infusion.Intravenous administration of AICAR runs into other problems not takeninto account in the animal and human trials to date. Adenosine, whendosed IV, has a half-life of around 12 seconds. This property is due tothe need to closely control extracellular adenosine levels. Bolus dosesof adenosine are taken up into erythrocytes and endothelial cellsproximal to the location of IV administration very rapidly. It can beassumed that a majority of acadesine given IV is likewise taken up byred blood cells and endothelial cells and is rapidly phosphorylated inthose cells to the active metabolite ZMP. Thus delivery to cells intissues other than blood, such as brain and muscle, is predicted to bevery inefficient.

Acadesine, once inside a cell and phosphorylated to ZMP, is ion-trappedinside the cell. ZMP will thus accumulate inside red blood cells andendothelial cells until it causes toxicity, rather than reaching othercell types where it may be needed. Therefore, IV administration of largedoses of the nucleoside acadesine will produce very small systemicexposure of the active species ZMP, along with toxic effects in RBCs andendothelial cells.

In order to achieve a desired therapeutic effect, however, it istypically necessary to deliver an active agent in a form that reachesother tissues where its effect is likely to be pharmaceuticallybeneficial rather than entering the first available cell. The presentinvention provides compounds of Formula (I) that, without being bound bytheory, are believed to act as prodrugs of ZMP, and are not rapidlyinternalized and trapped by the first cell they encounter as AICAR maybe, but are instead available systemically.

Once a compound of Formula (I) enters a cell, the prodrug moiety iscleaved, generating the active species ZMP. Accordingly, the compoundsof Formula (I) provide greater systemic bioavailability of the activemetabolite ZMP than AICAR itself does, while also reducing adverseeffects associated with quick entry into other cells, particularly redblood cells, that can be adversely affected by AICAR when it isadministered intravenously. These compounds of Formula (I) are thususeful to treat conditions treatable with acadesine or ZMP, but requirelower doses to be effective. Other features and advantages of theprodrugs of the invention, as well as methods of using them, will beappreciated in view of the following detailed descriptions.

DISCLOSURE OF THE INVENTION

The present invention relates to compounds of Formula (I),pharmaceutical compositions comprising these compounds, and methods touse these compounds and pharmaceutical compositions. In particular, theinvention provides methods to use the compounds of the invention fortreating disorders in brain and muscle tissues and the liver.

In one aspect, the present disclosure provides a heterocyclic compoundhaving a structure according to Formula (I):

wherein

-   -   R¹ is selected from:        -   (a) C₁-C₆ alkyl substituted with one or more groups selected            from list X;        -   (b) Q; and        -   (c) -L-Q;    -   R² is selected from:        -   (a) C₁-C₆ alkyl substituted with one or more groups selected            from List X;        -   (b) Q;        -   (c) -L-Q; and        -   (d) H;        -   Q is independently selected at each occurrence and            represents a ring selected from phenyl and a 5-6 membered            heteroaryl containing one to three heteroatoms selected from            N, O and S as ring members, and each Q is optionally            substituted with one to three groups selected from List M;        -   L is C₁-C₄ alkylene optionally substituted with one or two            groups selected from halo, oxo (═O), —OH, C₁-C₂ haloalkyl,            C₁-C₂ alkoxy, C₁-C₂ haloalkoxy, CN, COOR⁷, —OC(═O)R⁷, and            NR⁸R⁹;    -   R³ is H or —C(═O)—R⁶;    -   R⁴ is H or —C(═O)—R⁶;    -   R⁶ is H or C₁-C₆ alkyl that is optionally substituted with one        to three groups selected from halo, CN, hydroxy, C₁-C₄ alkoxy,        C₁-C₄ haloalkyl, —NR⁸R⁹, —OC(═O)—R⁷, and COOR⁷;    -   R⁷ is independently selected at each occurrence from H and C₁-C₆        alkyl optionally substituted with up to three groups selected        from halo, CN, hydroxy, C₁-C₄ alkoxy, and C₁-C₄ haloalkyl;    -   R⁸ and R⁹ are each independently selected at each occurrence        from H and C₁-C₄ alkyl optionally substituted with one or two        groups selected from List X;        -   or R⁸ and R⁹ taken together with the Nitrogen to which both            are attached form a 5-6 membered heterocyclic ring            optionally containing an additional heteroatom selected from            N, O and S as a ring member, and optionally substituted with            one to four groups selected from halo, oxo, C₁-C₂ alkyl,            hydroxy, C₁-C₂ alkoxy, CN, and COOR⁷;    -   R¹⁰ is independently at each occurrence C₁-C₆ alkyl optionally        substituted with up to three groups selected from halo, CN,        hydroxy, C₁-C₄ alkoxy, and C₁-C₄ haloalkyl;    -   List X consists of halo, CN, —OH, C₁-C₄ alkoxy, C₁-C₄ haloalkyl,        C₁-C₄ haloalkoxy, ═O, —COOR⁷, —OC(═O)R⁷, —O—COOR¹⁰, —SO₂R¹⁰,        —SO₂NR⁸R⁹, —O-Q, and —O-L-Q;    -   List M consists of halo, CN, NO₂, COOR⁷, CONR⁸R⁹, —SO₂R¹⁰,        —SO₂NR⁸R⁹, C₁-C₂ haloalkyl, C₁-C₂ haloalkoxy, C₁-C₂ alkoxy, and        C₁-C₂ alkyl;    -   or a pharmaceutically acceptable salt thereof.

In particular, the invention provides a compound selected from:

-   -   for use in treating indications as disclosed herein, especially        disorders in the muscle, brain and liver tissues, and for        preparation of medicaments useful to treat such indications.

The compounds of Formula (I) can be used to treat a condition for whichan AMP mimic is beneficial, especially in tissues other than blood,e.g., disorders in the muscles, brain and liver that are driven bybiochemical pathways that are regulated by AMP. As prodrugs of acadesinemonophosphate, the compounds of Formula (I) exhibit enhancedintracellular efficacy and systemicity compared to AICAR or ZMP, andprovide enhanced systemic delivery of ZMP. Thus the compounds of Formula(I) are useful to treat conditions in which AICAR and/or ZMP areeffective, and are more effective because the active agent is notdisproportionally trapped in blood cells. These conditions includemuscle disorders such as polymyositis, dermatomyositis, musculardystrophy, myasthenia gravis, amyotrophic lateral sclerosis,rhabdomyolysis, and cardiomyopathy, brain disorders includingAlzheimer's, stroke, cerebral atrophy, traumatic brain injury, anddementia, aging, diabetes, and cancers. While AICAR or ZMP has beenreported to be useful to treat acute lymphoblastic leukemia, B-cellchronic lymphocytic leukemia (B-CLL), type 2 diabetes, cardiac damage,and myocardial infarction, other B-cell lymphoproliferative disordersincluding splenic marginal zone lymphoma, mantle cell lymphoma,follicular lymphoma, lymphoplasmacytic lymphoma, and Waldenströmsyndrome, and to protect against ischemic injury during cardiac surgery,the invention provides methods to use the compounds of Formula (I),including pharmaceutically acceptable salts thereof, to treat muscledisorders such as polymyositis, dermatomyositis, muscular dystrophy,myasthenia gravis, amyotrophic lateral sclerosis, rhabdomyolysis, andcardiomyopathy, brain disorders including Alzheimer's, stroke, cerebralatrophy, traumatic brain injury, and dementia, aging, diabetes, andcancers

In one aspect, the invention provides compounds of Formula (I) asdescribed herein and their pharmaceutically acceptable salts fortreatment of the conditions mentioned above.

In some embodiments, the compounds described herein can be used intherapy, particularly to treat conditions mentioned above in subjects inneed of therapy for these conditions.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising a compound of Formula (I) as described hereinadmixed with at least one pharmaceutically acceptable carrier orexcipient to treat muscle disorders such as polymyositis,dermatomyositis, muscular dystrophy, myasthenia gravis, amyotrophiclateral sclerosis, rhabdomyolysis, and cardiomyopathy, brain disordersincluding Alzheimer's, stroke, cerebral atrophy, traumatic brain injury,and dementia, aging, diabetes, and cancers.

In yet another aspect, the disclosure provides a method for treatingand/or preventing conditions associated with insufficient activity of anAMP-regulated enzyme or pathway such as fructose-1,6-bisphosphatase,glycogen phosphorylase, phosphofructokinase-1, and AMPK. includingmuscle disorders such as polymyositis, dermatomyositis, musculardystrophy, myasthenia gravis, amyotrophic lateral sclerosis,rhabdomyolysis, and cardiomyopathy, brain disorders includingAlzheimer's, stroke, cerebral atrophy, traumatic brain injury, anddementia, aging, diabetes, and cancers.

In yet another aspect, the present disclosure provides for a use of acompound of Formula (I) as described herein for the manufacture of amedicament. In particular, the compounds are useful for manufacture of amedicament for treating conditions such as muscle disorders such aspolymyositis, dermatomyositis, muscular dystrophy, myasthenia gravis,amyotrophic lateral sclerosis, rhabdomyolysis, and cardiomyopathy, braindisorders including Alzheimer's, stroke, cerebral atrophy, traumaticbrain injury, and dementia, aging, diabetes, and cancers.

In yet another aspect, the present disclosure provides a combination fortreating and/or preventing a condition for which AICAR and/or ZMP haveefficacy, including those mentioned above. The combination comprises acompound of Formula (I) as described herein, and at least one additionaltherapeutic agent useful for treating the same subject to be treatedwith the compound of Formula (I), e.g., a subject having muscledisorders such as polymyositis, dermatomyositis, muscular dystrophy,myasthenia gravis, amyotrophic lateral sclerosis, rhabdomyolysis, andcardiomyopathy, brain disorders including Alzheimer's, stroke, cerebralatrophy, traumatic brain injury, and dementia, aging, diabetes, orcancer.

In yet another aspect, the present disclosure provides a method forproviding enhanced activation of PFK-1, FBP1, or GP.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the de novo purine biosynthetic pathway, including ZMPand AMP.

FIG. 2 depicts energy demand driving production of AMP. When the cellrequires more energy, ATP is quickly broken down into ADP. Adenylatekinase is an enzyme that transfers a phosphate from one ADP to another,thus synthesizing one ATP and one AMP. The ATP can be used for continuedenergy demands of the cell, while the AMP molecule acts as a low-energysignal to other cellular enzymes.

FIG. 3 illustrates AMP stimulation of glycolysis and ATP production.When the cell requires more energy, ATP breakdown, and AMP productionboth increase. AMP activates two key enzymes that mobilize storedglucose and promote glycolysis (the breakdown of glucose). Thispotentially increases ATP production, which can be used to fuel theenergy needs of the cell.

FIG. 4 illustrates the fructose-1,6-bisphosphatase role ingluconeogenesis. Fructose bisphosphatase catalyses the conversion offructose-1,6-bisphosphate to fructose-6-phosphate, which is the reverseof the reaction that is catalysed by phosphofructokinase in glycolysis.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entireties. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in a patent, application, or other publication thatis herein incorporated by reference, the definition set forth in thissection prevails over the definition incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more”.

The term “alkyl” as used herein refers to saturated hydrocarbon groupsin a straight, branched, or cyclic configuration or any combinationthereof, and particularly contemplated alkyl groups include those havingten or less carbon atoms, especially 1-6 carbon atoms and lower alkylgroups having 1-4 carbon atoms. Exemplary alkyl groups are methyl,ethyl, propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl,isopentyl, hexyl, cyclopropylmethyl, etc.

Alkyl groups can be unsubstituted, or they can be substituted to theextent that such substitution makes sense chemically. Typicalsubstituents include, but are not limited to, halo, ═O, ═N—CN,═N—OR^(a), ═NR^(a), —OR^(a), —NR^(a) ₂, —SR^(a), —SO₂R^(a), —SO₂NR^(a)₂, —NR^(a)SO₂R^(a), —NR^(a)CONR^(a) ₂, —NR^(a)COOR^(a), —NR^(a)COR^(a),—CN, —COOR^(a), —CONR^(a) ₂, —OOCR^(a), —COR^(a), and —NO₂, wherein eachR^(a) is independently H, C₁-C₄ alkyl, C₃-C₇ heterocyclyl, C₁-C₅ acyl,C₂-C₆ alkenyl, C₂-C₈ alkynyl, C₆-C₁₀ aryl, or C₅-C₁₀ heteroaryl, andeach R^(a) is optionally substituted with halo, ═O, ═N—CN, ═N—OR^(b),═NR^(b), OR^(b), NR^(b) ₂, SR^(b), SO₂R^(b), SO₂NR^(b) ₂,NR^(b)SO₂R^(b), NR^(b)CONR^(b) ₂, NR^(b)COOR^(b), NR^(b)COR^(b), CN,COOR^(b), CONR^(b) ₂, OOCR^(b), COR^(b), and NO₂, wherein each R^(b) isindependently H, C₁-C₄ alkyl, C₃-C₇ heterocyclyl, C₁-C₅ acyl, C₂-C₆alkenyl, C₂-C₈ alkynyl, C₆-C₁₀ aryl, or C₅-C₁₀ heteroaryl. Where asubstituent group contains two R^(a) or R^(b) groups on the same oradjacent atoms (e.g., —NR^(b) ₂, or —NR^(b)—C(O)—R^(b)), the two R^(a)or R^(b) groups can optionally be taken together with the atoms in thesubstituent group to which are attached to form a ring having 5-8 ringmembers, which can be substituted as allowed for the R^(a) or R^(b)itself, and can contain an additional heteroatom (N, O or S) as a ringmember.

The term “alkenyl” as used herein refers to an alkyl as defined abovehaving at least two carbon atoms and at least one carbon-carbon doublebond. Thus, particularly contemplated alkenyl groups include straight,branched, or cyclic alkenyl groups having two to ten carbon atoms (e.g.,ethenyl, propenyl, butenyl, pentenyl, etc.) or 5-10 atoms for cyclicalkenyl groups. Alkenyl groups are optionally substituted by groupssuitable for alkyl groups as set forth herein.

Similarly, the term “alkynyl” as used herein refers to an alkyl oralkenyl as defined above and having at least two (preferably three)carbon atoms and at least one carbon-carbon triple bond. Especiallycontemplated alkynyls include straight, branched, or cyclic alkyneshaving two to ten total carbon atoms (e.g., ethynyl, propynyl, butynyl,cyclopropylethynyl, etc.). Alkynyl groups are optionally substituted bygroups suitable for alkyl groups as set forth herein.

The term “cycloalkyl” as used herein refers to a cyclic alkane (i.e., inwhich a chain of carbon atoms of a hydrocarbon forms a ring), preferablyincluding three to eight carbon atoms. Thus, exemplary cycloalkanesinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,and cyclooctyl. Cycloalkyls can also include one or two double bonds,which form “cycloalkenyl” groups. Cycloalkyl groups are optionallysubstituted by groups suitable for alkyl groups as set forth herein.

The term “aryl” or “aromatic moiety” as used herein refers to anaromatic ring system. Thus, contemplated aryl groups include phenyl andnaphthyl. Furthermore, contemplated aryl groups may be fused (i.e.,covalently bound with 2 atoms on the first aromatic ring) with another5- or 6-membered heteroaryl, cycloalkyl, or heterocyclic group, and arethus termed “fused aryl” or “fused aromatic”.

Aromatic groups containing one or more heteroatoms (typically N, O or S)as ring members can be referred to as heteroaryl or heteroaromaticgroups. Typical heteroaromatic groups include monocyclic 5-6 memberedaromatic groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl,pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl, andimidazolyl and the fused bicyclic moieties formed by fusing one of thesemonocyclic groups with a phenyl ring or with any of the heteroaromaticmonocyclic groups to form an 8-10 membered bicyclic group such asindolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl,quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl,pyrazolopyrimidyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like.Any monocyclic or fused ring bicyclic system which has thecharacteristics of aromaticity in terms of electron distributionthroughout the ring system is included in this definition. It alsoincludes bicyclic groups where at least the ring which is directlyattached to the remainder of the molecule has the characteristics ofaromaticity. Typically, the aromatic ring systems contain 5-12 ringmember atoms.

As also used herein, the terms “heterocycle” and “heterocyclic” are usedto refer to any compound or radical in which a plurality of atoms form aring via a plurality of covalent bonds, wherein the ring includes atleast one atom other than a carbon atom as a ring member. Particularlycontemplated heterocyclic rings include 5- and 6-membered rings withnitrogen, sulfur, or oxygen as the non-carbon ring atom or atoms, e.g.,pyrrolidine, morpholine, piperidine, tetrahydrofuran, piperazine, andthe like. Typically these rings contain 0-1 oxygen or sulfur atoms, atleast one and typically 2-3 carbon atoms, and up to two nitrogen atomsas ring members. Further contemplated heterocycles may be fused (i.e.,covalently bound with two atoms on the first heterocyclic ring) to oneor two carbocyclic rings or heterocycles, and are thus termed “fusedheterocycle” or “fused heterocyclic ring” or “fused heterocyclicmoieties” as used herein. Where the fused ring is aromatic, these can bereferred to herein as ‘heteroaryl’ or heteroaromatic groups.

Heterocyclic groups that are not aromatic can be substituted with groupssuitable for alkyl group substituents, as set forth above, and also byC₁-C₆ alkyl groups.

Aryl and heteroaryl groups can be substituted where permitted. Suitablesubstituents include, but are not limited to, halo, —OR^(a), —NR^(a) ₂,—SR^(a), —SO₂R^(a), —SO₂NR^(a) ₂, —NR^(a)SO₂R^(a), —NR^(a)CONR^(a) ₂,—NR^(a)COOR^(a), —NR^(a)COR^(a), —CN, —COOR^(a), —CONR^(a) ₂, —OOCR^(a),—COR^(a), and —NO₂, wherein each R^(a) is independently H, C₁-C₄ alkyl,C₃-C₇ heterocyclyl, C₁-C₅ acyl, C₂-C₆ alkenyl, C₂-C₈ alkynyl, C₆-C₁₀aryl, or C₅-C₁₀ heteroaryl, and each R^(a) is optionally substitutedwith halo, ═O, ═N—CN, ═N—OR^(b), ═NR^(b), OR^(b), NR^(b) ₂, SR^(b),SO₂R^(b), SO₂NR^(b) ₂, NR^(b)SO₂R^(b), NR^(b)CONR^(b) ₂, NR^(b)COOR^(b),NR^(b)COR^(b), CN, COOR^(b), CONR^(b) ₂, OOCR^(b), COR^(b), and NO₂,wherein each R^(b) is independently H, C₁-C₄ alkyl, C₃-C₇ heterocyclyl,C₁-C₅ acyl, C₂-C₆ alkenyl, C₂-C₈ alkynyl, C₆-C₁₀ aryl, or C₅-C₁₀heteroaryl. Where a substituent group contains two R^(a) or R^(b) groupson the same or adjacent atoms (e.g., —NR^(b) ₂, or —NR^(b)—C(O)—R^(b)),the two R^(a) or R^(b) groups can optionally be taken together with theatoms in the substituent group to which are attached to form a ringhaving 5-8 ring members, which can be substituted as allowed for theR^(a) or R^(b) itself, and can contain an additional heteroatom (N, O orS) as a ring member.

The term “alkoxy” as used herein refers to a hydrocarbon group connectedthrough an oxygen atom, e.g., —O-Hc, wherein the hydrocarbon portion Hcmay have any number of carbon atoms, typically 1-10 carbon atoms, mayfurther include a double or triple bond and may include one or twooxygen, sulfur or nitrogen atoms in the alkyl chains, and can besubstituted with any of the groups disclosed herein as substituents foran alkyl group. For example, suitable alkoxy groups include methoxy,ethoxy, propyloxy, isopropoxy, methoxyethoxy, benzyloxy, allyloxy, andthe like. Similarly, the term “alkylthio” refers to alkylsulfides of thegeneral formula —S-Hc, wherein the hydrocarbon portion Hc is asdescribed for alkoxy groups. For example, contemplated alkylthio groupsinclude methylthio, ethylthio, isopropylthio, methoxyethylthio,benzylthio, allylthio, and the like.

The term ‘amino’ as used herein refers to the group —NH₂.

The term ‘acyl’ as used herein refers to a group of the formula—C(═O)-D, where D represents an alkyl, alkenyl, alkynyl, cycloalkyl,aryl, heteroaryl, or heterocycle as described above. Typical examplesare groups wherein D is a C₁-C₆ alkyl, C₂-C₆ alkenyl or alkynyl, orphenyl, each of which is optionally substituted. In some embodiments, Dcan be H, Me, Et, isopropyl, propyl, butyl, C₁-C₄ alkyl optionallysubstituted with —OH, —OMe, or NH₂, phenyl, halophenyl, alkylphenyl, andthe like.

The term “aryloxy” as used herein refers to an aryl group connecting toan oxygen atom, wherein the aryl group may be further substituted. Forexample, suitable aryloxy groups include phenyloxy, etc. Similarly, theterm “arylthio” as used herein refers to an aryl group connecting to asulfur atom, wherein the aryl group may be further substituted. Forexample, suitable arylthio groups include phenylthio, etc.

The hydrocarbon portion of each alkoxy, alkylthio, alkylamino, andaryloxy, etc. can be substituted as appropriate for the relevanthydrocarbon moiety.

The term “halogen” as used herein refers to fluorine, chlorine, bromineand iodine. Where present as a substituent group, halogen or halotypically refers to F or Cl or Br, more typically F or Cl.

The term “haloalkyl” refers to an alkyl group as described above,wherein one or more hydrogen atoms on the alkyl group have beensubstituted with a halo group. Examples of such groups include, withoutlimitation, fluoroalkyl groups, such as fluoroethyl, trifluoromethyl,difluoromethyl, trifluoroethyl and the like.

The term “haloalkoxy” refers to the group —O-haloalkyl and include, byway of example, groups such as trifluoromethoxy, and the like.

The term “substituted” as used herein refers to a replacement of ahydrogen atom of the unsubstituted group with at least one suitablesubstituent as described herein. Moreover, the term “substituted” alsoincludes multiple degrees of substitution, and where multiplesubstituents are disclosed or claimed, the substituted compound can beindependently substituted by one or more of the disclosed or claimedsubstituent moieties.

In addition to the disclosure herein, in a certain embodiment, a groupthat is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3substituents, 1 or 2 substituents, or 1 substituent.

It is understood that in all substituted groups defined above, compoundsarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups specifically contemplated herein are limited to substitutedaryl-(substituted aryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical or chemically unstable in an aqueous environment and/orsynthetically non-feasible. In addition, the subject compounds includeall stereochemical isomers arising from the substitution of thesecompounds, unless a specific isomer is disclosed. Where nucleosideanalogs are disclosed, the structures represent the specific enantiomerdepicted unless a mixture is indicated.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal, especiallya human (salts with counterions having acceptable mammalian safety for agiven dosage regime). Such salts can be derived from pharmaceuticallyacceptable inorganic or organic bases and from pharmaceuticallyacceptable inorganic or organic acids. “Pharmaceutically acceptablesalt” refers to pharmaceutically acceptable salts of a compound, many ofwhich are well known in the art. These salts are derived from a varietyof organic and inorganic counter ions well known in the art and include,by way of example only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, formate, tartrate, besylate, mesylate,acetate, maleate, oxalate, and the like.

The term “salt thereof” refers to a compound formed when a neutralorganic compound is protonated or deprotonated to produce an ionicorganic moiety that is associated with an oppositely-charged counterion.For example, this may refer to a salt formed when a proton of an acidicorganic molecule is replaced by a cation, such as a metal cation or anorganic cation. As another example, salts of the present compoundsinclude those wherein the compound is protonated by an inorganic ororganic acid to form a cation, with the conjugate base of the inorganicor organic acid as the anionic component of the salt. Where applicable,the salt is a pharmaceutically acceptable salt, although this is notrequired for salts of compounds that are not intended for administrationto a patient, such as precursors to the final drug compound orintermediates useful for synthesis of a compound of Formula (I).

The compounds and compositions described herein can be administered to asubject in need of treatment for any of the conditions disclosed above.The subject is typically a mammal diagnosed as being in need oftreatment for one or more of such disorders, and preferably the subjectis a human. The methods comprise administering an effective amount of atleast one compound of Formula (I), optionally in the form of apharmaceutical composition. Optionally, the compound may be administeredin combination with one or more additional therapeutic agents,particularly therapeutic agents useful for treating the conditionafflicting the particular subject.

Exemplary Embodiments

The following enumerated embodiments are representative of theinvention:

In some embodiments, the present disclosure provides compound SB00039,compound 5 or compound 7, and the pharmaceutically acceptable saltsthereof, and methods of treatment and pharmaceutical compositions usingthese compounds.

Pharmaceutical Compositions

The compounds of the invention can be prepared and administered aspharmaceutical compositions comprising a compound of Formula (I) admixedwith at least one pharmaceutically acceptable carrier or excipient. Insome embodiments, the pharmaceutical composition comprises at least twopharmaceutically acceptable carrier or excipient components. Suitablecarriers include water, optionally buffered such as with a phosphate,carbonate, acetate, or similar buffering agent, and potentially also atleast one solvating component such as a co-solvent or cyclodextrin.Suitable carriers are disclosed in the following formulations.

In some embodiments, the compounds of Formula (I) are provided in a formsuitable for dissolution or suspension in an acceptable intravenousform, thus compounds of Formula (I) may be combined with one or morecarrier or excipient components, and optionally then lyophilized orotherwise concentrated to a form that can readily be reconstituted withan aqueous carrier such as saline, phosphate-buffered saline, glucose,lactate or Ringer's lactate, typically at an isotonic concentrationlevel, for intravenous administration, including infusion.

Formulations

Any suitable formulation of the compounds described herein can beprepared. See generally, Remington's Pharmaceutical Sciences, (2000)Hoover, J. E. editor, 20th edition, Lippincott Williams and WilkinsPublishing Company, Easton, Pa., pages 780-857. A formulation isselected to be suitable for an appropriate route of administration. Incases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compounds as saltsmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids that form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts. Pharmaceutically acceptable salts are obtainedusing standard procedures well known in the art, for example, by asufficiently basic compound such as an amine with a suitable acid,affording a physiologically acceptable anion. Alkali metal (e.g.,sodium, potassium or lithium) or alkaline earth metal (e.g., calcium)salts of carboxylic acids also are made.

Where compounds of Formula (I) are administered in a pharmacologicalcomposition, it is contemplated that the compounds can be formulated inadmixture with a pharmaceutically acceptable excipient and/or carrier.For example, contemplated compounds can be administered orally asneutral compounds or as pharmaceutically acceptable salts, orintravenously in a physiological saline solution or similar suitableisotonic solution. Conventional buffers such as phosphates, bicarbonatesor citrates can be used for this purpose. Of course, one of ordinaryskill in the art may modify the formulations within the teachings of thespecification to provide numerous formulations for a particular route ofadministration. In particular, contemplated compounds may be modified torender them more soluble in water or other vehicle, which for example,may be easily accomplished with minor modifications (salt formulation,esterification, etc.) that are well within the ordinary skill in theart. It is also well within the ordinary skill of the art to modify theroute of administration and dosage regimen of a particular compound inorder to manage the pharmacokinetics of the present compounds formaximum beneficial effect in a patient.

The compounds of Formula I as described herein are generally soluble inorganic solvents such as chloroform, dichloromethane, ethyl acetate,ethanol, methanol, isopropanol, acetonitrile, glycerol,N,N-dimethylformamide, N,N-dimetheylaceatmide, dimethylsulfoxide, etc.In one embodiment, the present invention provides formulations preparedby mixing a compound of Formula I with a pharmaceutically acceptablecarrier. In one aspect, the formulation may be prepared using a methodcomprising: a) dissolving a described compound in a water-solubleorganic solvent, a non-ionic solvent, a water-soluble lipid, acyclodextrin, a vitamin such as tocopherol, a fatty acid, a fatty acidester, a phospholipid, or a combination thereof, to provide a solution;and b) adding saline or a buffer containing 1-10% carbohydrate solution.In one example, the carbohydrate comprises dextrose. The pharmaceuticalcompositions obtained using the present methods are stable and usefulfor animal and clinical applications.

Illustrative examples of water soluble organic solvents for use in thepresent compositions and methods include and are not limited topolyethylene glycol (PEG), alcohols, acetonitrile,N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide,dimethyl sulfoxide, or a combination thereof. Examples of alcoholsinclude but are not limited to methanol, ethanol, isopropanol, glycerol,or propylene glycol.

Illustrative examples of water soluble non-ionic surfactants for use inthe present methods and compositions include and are not limited toCREMOPHOR® EL, polyethylene glycol modified CREMOPHOR®(polyoxyethyleneglyceroltriricinoleat 35), hydrogenated CREMOPHOR® RH40,hydrogenated CREMOPHOR® RH60, PEG-succinate, polysorbate 20, polysorbate80, SOLUTOL® HS (polyethylene glycol 660 12-hydroxystearate), sorbitanmonooleate, poloxamer, LABRAFIL® (ethoxylated persic oil), LABRASOL®(capryl-caproyl macrogol-8-glyceride), GELUCIRE® (glycerol ester),SOFTIGEN® (PEG 6 caprylic glyceride), glycerin, glycol-polysorbate, or acombination thereof.

Illustrative examples of water soluble lipids for use in the presentmethods and compositions include but are not limited to vegetable oils,triglycerides, plant oils, or a combination thereof. Examples of lipidoils include but are not limited to castor oil, polyoxyl castor oil,corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil,safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil,hydrogenated soybean oil, a triglyceride of coconut oil, palm seed oil,and hydrogenated forms thereof, or a combination thereof.

Illustrative examples of fatty acids and fatty acid esters for use inthe present methods and compositions include but are not limited tooleic acid, monoglycerides, diglycerides, a mono- or di-fatty acid esterof PEG, or a combination thereof. Illustrative examples of cyclodextrinsfor use in the present methods include but are not limited toalpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin,or sulfobutyl ether-beta-cyclodextrin.

Illustrative examples of phospholipids for use in the present methodsand compositions include but are not limited to soy phosphatidylcholine,or distearoyl phosphatidylglycerol, and hydrogenated forms thereof, or acombination thereof.

In addition to carriers useful in the pharmaceutical compositions of theinvention, compounds may be admixed with excipients beneficial tosolubilize, stabilize, or otherwise modify the compound of Formula (I)to provide a suitable formulated composition for storage oradministration. Excipients include colorants, flavoring agents,stabilizers, disintegrants, glidants, lubricants, preservatives and thelike.

In many embodiments, the compounds of Formula (I) are formulated assolutions, emulsions, dispersions, or suspensions that are suitable forinjection or infusion, or formulated so they can readily be diluted forintravenous administration or infusion.

One of ordinary skill in the art may modify the formulations within theteachings of the specification to provide numerous formulations for aparticular route of administration. In particular, the compounds may bemodified to render them more soluble in water or other carriers, oftenusing solvating agents disclosed above, particularly ones that are knownin the art to be pharmaceutically acceptable and suited foradministration via injection or infusion. It is also well within theordinary skill of the art to select or modify the route ofadministration and dosage regimen of a particular compound in order tomanage the pharmacokinetics of the present compounds for maximumbeneficial effect in a patient.

Drug Combinations

The methods of using compounds of Formula (I) comprise administering aneffective amount of at least one exemplary compound of Formula (I);optionally the compound may be administered in combination with one ormore additional therapeutic agents, particularly at least onetherapeutic agent that is useful for treatment of the same condition forwhich the compound of Formula (I) is indicated, or treatment of symptomsor complications of this condition, or reduction of side effectsassociated with treatment of this condition.

The additional therapeutic agent(s) may be administered in a separatepharmaceutical composition from the compound of Formula (I), or may beincluded with the compound of the present disclosure in a singlepharmaceutical composition. The additional therapeutic agent may beadministered simultaneously with, prior to, or after administration ofthe compound(s) of Formula (I) according to the present disclosure.

Methods of Using Compounds of Formula (I) and PharmaceuticalCompositions Thereof

The present invention provides pharmaceutical compositions for thetreatment and/or prevention of conditions described herein, and methodsof using these compounds and compositions containing a compound ofFormula I as described herein. The methods typically compriseadministering the compound or composition to a subject, typically ahuman. In some embodiments, the method is for treatment and the subjectis one already having been diagnosed as in need of such treatment for acondition selected from those described herein. Preferably, the compoundor composition is administered in an amount effective to treat thesubject's condition. Selection of a suitable compound of Formula (I),and suitable formulation thereof, and suitable routes of administrationand dosage therefor are within the ordinary level of skill in the art inview of the disclosures provided herein and conventional testing,treatment and monitoring practices. For solubility reasons, the compoundof Formula I is frequently formulated and administered as apharmaceutically acceptable salt.

To practice the method of the present invention, compounds havingformula (I) and pharmaceutical compositions thereof may be administeredorally, parenterally, by inhalation, topically, rectally, nasally,buccally, vaginally, via an implanted reservoir, or other drugadministration methods. Typically, compounds of Formula (I) areadministered by parenteral routes. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional and intracranial injection or infusion techniques. Apreferred route of administration for compounds of Formula (I) isintravenous injection or intravenous infusion, commonly in a solution orsuspension comprising a typical IV fluid such as isotonic saline orisotonic glucose solution or Ringer's lactate.

Suitable carriers and other pharmaceutical composition components aretypically sterile. A sterile injectable composition, such as a sterileinjectable aqueous or oleaginous suspension, may be formulated accordingto techniques known in the art using suitable dispersing or wettingagents and suspending agents as needed. The sterile injectablepreparation may also be a sterile injectable solution or suspension in anontoxic parenterally acceptable diluent or solvent. Among theacceptable vehicles and solvents that may be employed include mannitol,dextrose, citrate buffer, water, Ringer's solution and isotonic sodiumchloride solution.

In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium (e.g., synthetic mono- or diglycerides).Fatty acids, such as oleic acid and its glyceride derivatives, areuseful in the preparation of injectables, as are pharmaceuticallyacceptable oils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions can alsocontain a long-chain alcohol diluent or dispersant, or carboxymethylcellulose or similar dispersing agents. Various emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms canalso be used for the purpose of formulation.

A composition for oral administration may be any orally acceptabledosage form including, but not limited to, tablets, capsules, emulsionsand aqueous suspensions, dispersions and solutions. In the case oftablets for oral use, commonly used carriers include lactose and cornstarch. Lubricating agents, such as magnesium stearate, can also beadded. For oral administration in a capsule form, useful diluentsinclude lactose and dried corn starch. When aqueous suspensions oremulsions are administered orally, the active ingredient can besuspended or dissolved in an oily phase combined with emulsifying orsuspending agents. If needed, certain sweetening, flavoring, or coloringagents can be added. A nasal aerosol or inhalation compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation and can be prepared as solutions in, for example saline,employing suitable preservatives (for example, benzyl alcohol),absorption promoters to enhance bioavailability, and/or othersolubilizing or dispersing agents known in the art.

Synthesis of Compounds of Formula (I)

Compound 1: The starting nucleoside was (0.166 g, 0.643 mmol) wassuspended in trimethylphosphate (3 ml). With stirring under nitrogen,neat phosphorous oxychloride (0.359 ml, 3.86 mmol) was added. Thereaction was stirred for 2 hours at room temperature. Methanol (0.500ml, 12 mmol) was then added and the reaction was stirred for 30 minutesat room temperature. Triethylamine (2 ml, 15 mmol), was carefully addedto quench. The reaction was diluted with acetonitrile 40 ml and loadedonto a silica gel column. The desired product was eluted using agradient of 0 to 30% methanol in acetonitrile. (0.075 g, 32%) ([M+1],367)

Compound 2: The starting nucleoside (0.155 g, 0.6 mmol) was suspended intrimethylphosphate (3 ml). With stirring under nitrogen, phosphorousoxychloride (0.335 ml, 3.6 mmol) was added neat. The reaction wasstirred for 2 hours at room temperature. 4-nitrophenol (2.085 g, 15mmol) was then added and the reaction was stirred for 30 minutes at roomtemperature. Triethylamine (15 mmol, 2 ml) was carefully added toquench. The reaction was diluted with ethyl acetate (30 ml) and loadedonto a silica gel column. The desired product was eluted using agradient of 0 to 10% methanol in ethyl acetate. (0.174 g, 50%) ([M+1],581)

Compound 3: The starting di-(nitrophenyl) phosphate nucleoside (0.100 g,0.17 mmol) was dissolved in methanol (15 ml). With stirring undernitrogen, triethylamine (0.117 ml, 0.85 mmol) was added neat. Thereaction was stirred for 3 hours at room temperature. Acetic acid (0.120ml, 2 mmol) was added to quench. Solvents were removed by rotaryevaporation. The residue was dissolved in DCM and loaded onto a silicagel column. The desired product was eluted using a gradient of 0 to 20%methanol in ethyl acetate. (0.035 g, 43%) ([M+1], 474)

Compound 4: The starting nitrophenylmethyl phosphate nucleoside (0.025g, 0.05 mmol) was dissolved in a 1:1 mixture of THF and water (5 ml).With stirring under nitrogen, triethylamine (0.034 ml, 0.25 mmol) wasadded neat. The reaction was heated to 55° C. and stirred for 2 hours.Acetic acid (0.050 ml, 0.8 mmol) was added to quench. Solvents wereremoved by rotary evaporation. The desired product was purified by prephplc (0.007 g, 40%) ([M+1], 353)

Compound 5: The starting nucleoside (0.100 g, 0.39 mmol) was suspendedin trimethylphosphate (3 ml). With stirring under nitrogen, protonsponge (0.332 g, 1.55 mmol) was added. The reaction was stirred for 10minutes at room temperature and then 4-chlorophenylphosphorodichloridate(0.252 ml, 1.55 mmol) was added neat. The reaction was stirred for 1hour at room temperature. Water (1 ml) was then added and the reactionwas stirred for 30 minutes at room temperature. Triethylamine (10 mmol,2 ml) was carefully added to quench. The reaction was diluted withacetonitrile (30 ml) and loaded onto a silica gel column. The desiredproduct was eluted using a gradient of 0 to 30% methanol inacetonitrile. (0.070 g, 40%) ([M+1], 449)

Compound 6: The starting nucleoside (0.204 g, 0.79 mmol) and thephosphate TEA salt (0.514 g, 1.58 mmol) were suspended in acetonitrile(10 ml). With stirring under nitrogen, 1-methylimidazole (0.314 ml, 3.95mmol) was added. The reaction was stirred for 10 minutes at roomtemperature and then bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.402g, 1.58 mmol) was added. The reaction was stirred for 1 hour at roomtemperature then additional bis(2-oxo-3-oxazolidinyl)phosphinic chloride(0.804 g, 3.16 mmol) was added. The reaction was stirred for 2 hours.When the reaction was complete by LC/MS, solvents were removed by rotaryevaporation. The desired product was purified by preparatory HPLC.(0.075 g, 17%) ([M+1], 571)

Compound 7: The starting poc phosphate nucleoside (0.050 g, 0.09 mmol)was dissolved in a 1:1 mixture of THF and water (4 ml). With stirringunder nitrogen, triethylamine (0.027 ml, 0.2 mmol) was added neat. Thereaction was stirred for 1 hour at room temperature. When the reactionwas complete by LC/MS, acetic acid (0.100 ml, 1.7 mmol) was added toquench. Solvents were removed by rotary evaporation. The desired productwas purified by prep hplc (0.013 g, 32%) ([M+1], 455)

Biological Activity of Compounds of Formula (I)

Some exemplary assays and examples for assessing therapeutic efficacy,e.g., anticancer effects, of exemplary compounds of the invention aredescribed herein or well known in the art. HEK293 is a human kidney cellline, and efficacy on HEK293 is considered to be predictive ofusefulness for therapeutic treatment. In the Figures below, the activityof the compounds on HEK293 cells according to the method described belowis reported as the CC50, or 50% cytotoxic concentration (micromolar).

CC50 in HEK293 cells

The first two structures above are the known compounds AICAR and ZMP.The third structure (SB00024) is an uncleavable analogue of ZMP (one notexpected to readily cleave in vivo), and was inactive at 500 uM. Thefourth structure (SB00039) is an exemplary prodrug of Formula I, andexhibits roughly 100 times the potency of AICAR. The final structure(SB00038) is the byproduct of the unmasking of the prodrug. This wastested to show that the potency of SB00039 comes from the prodrugpenetrating the cell and being unmasked to produce ZMP, and not from thebyproduct.

The potency of SM00039 is surprising, because other prodrugs of ZMP didnot exhibit improved potency. FIG. 2 shows a number of other prodrugs ofZMP that did not enhance its potency in the HEK239 assay.

A few ZMP prodrugs have been reported in the literature, but they alsodo not show efficacy in the HEK293 assay.

McGuigan, Eur. J. Med. Chem. 70, 326-40 (2013).

Bookser, et. al, J. Combinatorial Chem. 10(4), 567-72 (2008). Materialand Methods Cell Culture and Reagents

HEK293 cells were obtained from American Type Culture Collection (ATCC)(Rockville Md.). Cells were maintained in Dulbecco's modified Eagle'smedium (DMEM) (Invitrogen, Carlsbad, Calif.) with 10% fetal bovine serum(FBS), 10 mM HEPES and 1 mM sodium pyruvate. Cells were grown at 37° C.in a humidified incubator with a gas phase of 5% CO₂. Cells were grownfor 24 hours in a Tripleflask (NUNC) to −95% confluence and thenresuspended for dispensing at 125,000 cells/mL of DMEM, 10% FBS, 10 mMHepes and 1 mM sodium pyruvate.

HEK293 Cytotoxicity Assay

HEK293 cells were seeded in 96 well microtiter plates at 10000cells/well. After 24 hours of incubation an equal volume of fresh mediacontaining test compounds at given concentrations was added. Plates werethen incubated and cells were allowed to proliferate for 48 hours at 37°C., 95% humidity, and 5% CO₂. At 72 hours, plates were removed from theincubator and cooled for 15 minutes to room temperature. A solution ofPromega CellTiterGlo (100 microliters) was added by Thermo Combi and theplates were allowed to sit for 10 minutes before reading on aPerkin-Elmer EnVision with US LUM settings for 0.1 sec per well. The 50%cytotoxicity concentration (CC50) was defined as the compoundconcentration required to reduce the viable cells by 50%.

The detailed description set forth above is provided to aid thoseskilled in the art in practicing the present invention. However, theinvention described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description which do not depart from thespirit or scope of the present inventive discovery. Such modificationsare also intended to fall within the scope of the appended claims.

All publications, patents, patent applications and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentinvention.

1. A method to treat a brain disorder, muscle disorder, liver disorder,diabetes or cancer, wherein the method comprises administering acompound of Formula (I):

wherein: R¹ is selected from: (a) C₁-C₆ alkyl substituted with one ormore groups selected from list X; (b) Q; and (c) -L-Q; R² is selectedfrom: (a) H; (b) Q; and (c) -L-Q; and Q is independently selected ateach occurrence and represents a ring selected from phenyl and a 5-6membered heteroaryl containing one to three heteroatoms selected from N,O and S as ring members, and each Q is optionally substituted with oneto three groups selected from List M; L is C₁-C₄ alkylene optionallysubstituted with one or two groups selected from halo, oxo (═O), —OH,C₁-C₂ haloalkyl, C₁-C₂ alkoxy, C₁-C₂ haloalkoxy, CN, COOR⁷, —OC(═O)R⁷,and NR⁸R⁹; R³ is H or —C(═O)—R⁶; R⁴ is H or —C(═O)—R⁶; R⁶ is H or C₁-C₆alkyl that is optionally substituted with one to three groups selectedfrom halo, CN, hydroxy, C₁-C₄ alkoxy, C₁-C₄ haloalkyl, —NR⁸R⁹,—OC(═O)—R⁷, and COOR⁷; R⁷ is independently selected at each occurrencefrom H and C₁-C₆ alkyl optionally substituted with up to three groupsselected from halo, CN, hydroxy, C₁-C₄ alkoxy, and C₁-C₄ haloalkyl; R⁸and R⁹ are each independently selected at each occurrence from H andC₁-C₄ alkyl optionally substituted with one or two groups selected fromList X; or R⁸ and R⁹ taken together with the Nitrogen to which both areattached form a 5-6 membered heterocyclic ring optionally containing anadditional heteroatom selected from N, O and S as a ring member, andoptionally substituted with one to four groups selected from halo, oxo,C₁-C₂ alkyl, hydroxy, C₁-C₂ alkoxy, CN, and COOR⁷; R¹⁰ is independentlyat each occurrence C₁-C₆ alkyl optionally substituted with up to threegroups selected from halo, CN, hydroxy, C₁-C₄ alkoxy, and C₁-C₄haloalkyl; List X consists of halo, CN, —OH, C₁-C₄ alkoxy, C₁-C₄haloalkyl, C₁-C₄ haloalkoxy, ═O, —COOR⁷, —OC(═O)R⁷, —O—COOR¹⁰, —SO₂R¹⁰,—SO₂NR⁸R⁹, —O-Q, and —O-L-Q; List M consists of halo, CN, NO₂, COOR⁷,CONR⁸R⁹, —SO₂R¹⁰, —SO₂NR⁸R⁹, C₁-C₂ haloalkyl, C₁-C₂ haloalkoxy, C₁-C₂alkoxy, and C₁-C₂ alkyl; or a pharmaceutically acceptable salt thereof,to a subject in need of such treatment.
 2. The method of claim 1,wherein R¹ is C₁-C₄ alkyl substituted with one to three groups selectedfrom list X; or a pharmaceutically acceptable salt thereof.
 3. Themethod of claim 1, wherein R¹ is —CH₂—O—C(═O)—OR¹⁰, where R¹⁰ is C₁-C₄alkyl optionally substituted with C₁-C₂ alkoxy, COOR⁷, or CN, or apharmaceutically acceptable salt thereof.
 4. The method of claim 1,wherein R¹ is phenyl, optionally substituted with one to three groupsselected from List M, or a pharmaceutically acceptable salt thereof. 5.The method of claim 1, wherein R² is H, or a pharmaceutically acceptablesalt thereof.
 6. The method of claim 1, wherein R² is Q, or apharmaceutically acceptable salt thereof.
 7. The method of claim 1,wherein R³ is H, or a pharmaceutically acceptable salt thereof.
 8. Themethod of claim 1, wherein R⁴ is H, or a pharmaceutically acceptablesalt thereof.
 9. The method of claim 1, wherein R³ and R⁴ are different;or a pharmaceutically acceptable salt thereof.
 10. The method accordingto claim 1, wherein R¹ is selected from a nitrophenyl group, ahalophenyl group, and a group of the formula —CH₂—OC(═O)—O—(C₁-C₄alkyl), and R² is selected from H, a nitrophenyl group, and a halophenylgroup; or a pharmaceutically acceptable salt thereof.
 11. The method ofclaim 1, wherein the compound is selected from

or a pharmaceutically acceptable salt thereof.
 12. The method of claim1, wherein the condition is a brain condition selected from Alzheimer's,stroke, cerebral atrophy, traumatic brain injury, and dementia, muscledisorders such as polymyositis, dermatomyositis, muscular dystrophy,myasthenia gravis, amyotrophic lateral sclerosis, rhabdomyolysis, andcardiomyopathy, brain disorders including Alzheimer's, stroke, cerebralatrophy, traumatic brain injury, and dementia, aging, diabetes, orcancer.
 13. The method of claim 1, wherein the condition is a muscledisorder selected from polymyositis, dermatomyositis, musculardystrophy, myasthenia gravis, amyotrophic lateral sclerosis,rhabdomyolysis, and cardiomyopathy.
 14. The method of claim 1, whereinthe condition is a liver disorder.
 15. The method of claim 1, whereinthe compound is of the formula

or a pharmaceutically acceptable salt thereof.
 16. A method to activatefructose-1,6-bisphosphatase, glycogen phosphorylase, orphosphofructokinase-1 in a cell, which comprises contacting the cellwith a compound according to claim 1.